Compounds and methods for labeling oligonucleotides

ABSTRACT

A compound having the general formula shown below: 
                         
where R 1-6  are independently selected from the group consisting of an electron withdrawing group, an alkyl group, an aryl group, hydrogen, a heteroaryl group, and a five or six member ring structure formed from the R 1  and R 2  pair, the R 3  and R 4  pair, the R 4  and R 5  pair, or the R 5  and R 6  pair; R 7  is a substituted or unsubstituted aryl group; and Y is a nucleophile.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/623,811filed Nov. 23, 2009, which is a divisional of Ser. No. 12/535,220, filedAug. 4, 2009, now U.S. Pat. No. 7,645,872, which is a divisional of U.S.application Ser. No. 12/352,125, filed Jan. 12, 2009, now U.S. Pat. No.7,605,243, which is a divisional of U.S. application Ser. No.11/438,606, filed May 22, 2006, now U.S. Pat. No. 7,476,735, whichclaims the priority benefit of U.S. Provisional Application No.60/683,278, filed May 20, 2005. These applications are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

This invention pertains to compounds and methods for labelingoligonucleotides. The invention also provides kits that contain at leastone of the disclosed compounds.

BACKGROUND OF THE INVENTION

Oligonucleotides are often modified or labeled with reporter moietiessuch as quenchers, fluorophores, biotin, etc. These labeledoligonucleotides can provide information regarding binding and otherbiological phenomena, the structure of DNA, the association ofmacromolecules, and the size and mobility of protein and DNA complexes.

Several attachment chemistries are currently used for modifyingoligonucleotides. For example, primary amino groups are widely used toattach modifiers, reporter moieties or labels to an oligonucleotide. Inaddition, they can be used to attach an oligonucleotide to a solidsurface.

Stable Schiff base linkers have been used for the synthesis of labeledoligonucleotides. (Dey & Sheppard (2001) Org. Lett. Vol. 3,25:3983-3986, which is incorporated herein by reference). The methodshave been limited to the post-synthetic attachment of labels, and theproposed methods have not been commercially viable alternatives tostandard synthesis approaches. Previously described post-syntheticmethods permit the incorporation of only a single type of reportermoiety or multiple copies of the same reporter moiety into anoligonucleotide.

Labeled oligonucleotides have a wide variety of useful applications. Forexample, light quenching processes that rely on the interaction of afluorophore and quencher as their spatial relationship changes can beused in convenient processes for detecting and/or identifyingoligonucleotides and other biological phenomena. In one such method, thechange in fluorescence of a fluorophore or quencher can be monitored astwo oligonucleotides (one containing a fluorophore and one containing aquencher) hybridize to each other. The hybridization can be detectedwithout intervening purification steps that separate unhybridized fromhybridized oligonucleotides. Currently, quencher groups are commonlyplaced at the end of a probe sequence while the fluorophore is placed atthe opposite end, solely for ease of synthesis. However, in someapplications, such as real-time PCR, dual-labeled probes are moreeffective when the labels are placed closer to each other.

Perhaps the most common mechanism of fluorescent quenching isfluorescent resonance energy transfer (“FRET”). For FRET to occur, afluorophore and a fluorescent quencher must be within a suitabledistance for the quencher to absorb energy from the donor. In addition,there must be overlap between the emission spectrum of the fluorescentdonor and the absorbance spectrum of the quencher. This requirementcomplicates the design of probes that utilize FRET because not allpotential quencher/fluorophore pairs can be used. For example, thequencher known as BHQ-1, which absorbs light in the wavelength range ofabout 520-550 nm, can quench the fluorescent light emitted from thefluorophore, fluorescein, which fluoresces maximally at about 520 nm. Incontrast, the quencher BHQ-3, which absorbs light in the wavelengthrange of about 650-700 nm would be almost completely ineffective atquenching the fluorescence of fluorescein through FRET but would bequite effective at quenching the fluorescence of the fluorophore knownas Cy5 which fluoresces at about 670 nm.

Oligonucleotides labeled with fluorophores and quenchers can also beused to monitor the kinetics of PCR amplification. For example, a PCRreaction is performed using oligonucleotides designed to hybridize tothe 3′ side (“downstream”) of an amplification primer so that the 5′-3′exonuclease activity of a polymerase digests the 5′ end of the probe,cleaving off one of the dyes. The fluorescence intensity of the sampleincreases and can be monitored as the probe is digested during thecourse of amplification.

Similar oligonucleotide compositions may be used in othermolecular/cellular biology and diagnostic assays, such as end-point PCR,in situ hybridizations, in vivo DNA and RNA species detection, singlenucleotide polymorphism (SNPs) analysis, enzyme assays, and in vivo andin vitro whole cell assays.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method for linking a reporter moiety to anoligonucleotide comprising reacting a reporter moiety having an oximeforming nucleophile substituent with an oxo substituted reactant coupledto a solid support to form an oxime bond between the reporter moiety andthe reactant. The reporter moieties include, but are not limited to,quenchers, fluorophores, biotin, digoxigenin, peptides and proteins. Theinvention also provides an oligonucleotide labeled with at least twodifferent reporter moieties.

This invention further provides novel azo quenchers having the generalformula shown below in Formula (I):

Each of R₁₋₆ is individually selected from the group consisting ofhydrogen; electron withdrawing groups such as halogens, NO₂, SO₃R_(S),SO₂N(R_(N))₂, CN, CNS, keto, alkoxy groups; C₁-C₁₀ alkyl groups; arylgroups; and heteroaryl groups. R_(N) and R_(S) can be C₁-C₁₀ alkylgroups, which may be saturated or unsaturated, branched or unbranched,and substituted or unsubstituted, or aryl groups, which may besubstituted or unsubstituted. Suitable substituents include electronwithdrawing groups, such as those described above.

R₇ can be any aryl group that can be joined to the conjugated ringsystem by an azo bond to form a compound that is capable of quenchingthe fluorescence of a fluorophore. Suitable aryl groups include phenyl,naphthyl, benzyl, xylyl, toluoyl, pyridyl and anilinyl, among othergroups. R₇ can be substituted or derivatized with at least one linkinggroup for linking the quencher compound to other compounds of interest.

Y is a nucleophile-containing group capable of reacting with an oxogroup to form an oxime bond, such as aminooxy or hydrazine. In addition,the R₁/R₂ pair, R₃/R₄ pair, R₄/R₅ pair and R₅/R₆ pair can be combined toform ring structures having five or six ring members. These ringstructures can be substituted with hydrogen, heteroatom-substitutedalkyl, halogen, alkenyl, alkoxy, alkoxy-alkyl, hydroxyl,trifluoromethyl, cyano, nitro, acyl, acyloxy, amino, alkylamino,dialkylamino, carboxyl, carbalkoxyl, carboxamido, mercapto, sulfamoyl,phenyl, and napthyl.

In addition, this invention provides an oligonucleotide labeled with thenovel quencher as well as a method of detecting hybridization ofoligonucleotides using the labeled oligonucleotide.

The invention provides compositions comprising a quencher linked to acompound selected from the group consisting of an antigen, a steroid, avitamin, a drug, a hapten, a metabolite, a toxin, an environmentalpollutant, an amino acid, a protein, a carbohydrate, a solid support, alinker, and a lipid, wherein the quencher is attached to the compoundvia an oxime bond. The invention further provides compositionscomprising labeled oligonucleotides and solid supports. The inventionalso provides kits comprising at least one composition of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of a compound of Formula (I).

FIG. 2 shows the synthesis of a ketone phosphoramidite.

FIG. 3 shows the synthesis of aminooxy conjugated controlled pore glasssupports.

FIG. 4 shows the introduction of the aminooxy group into a reportermoiety that is stable to basic conditions.

FIG. 5 shows the introduction of the aminooxy group into a base labilereporter moiety.

FIG. 6 shows real-time PCR data for Probe SEQ ID NO: 1 in amulticomponent view. Fluorescein data plot is positioned as the firstcurve in the upper graph and represents signal from the probe. Rox dataplot is positioned as the second (flat) curve in the upper plot andrepresents detection control. Temperature trace during thermal cyclingis plotted in the lower graph.

FIG. 7 shows real-time PCR data for Probe SEQ ID NO: 1 as amplificationtraces. Reactions were done using input target amounts of 5×10⁶molecules, 5×10⁴ molecules, and 5×10² molecules which are shown left toright. All target concentrations were run in triplicate.

FIG. 8 shows real-time PCR amplification traces for Probe SEQ ID NO: 2.Reactions were done using input target amounts of 5×10⁶ molecules, 5×10⁴molecules, and 5×10² molecules which are shown left to right. All targetconcentrations were run in triplicate.

FIG. 9 shows real-time PCR amplification traces for Probe SEQ ID NO: 3.Reactions were done using input target amounts of 5×10⁶ molecules, 5×10⁴molecules, and 5×10² molecules which are shown left to right. All targetconcentrations were run in triplicate.

FIG. 10 shows real-time PCR amplification traces for Probe SEQ ID NO: 4.Reactions were done using input target amounts of 5×10⁶ molecules, 5×10⁴molecules, and 5×10² molecules which are shown left to right. All targetconcentrations were run in triplicate.

FIG. 11 shows real-time PCR amplification traces for Probe SEQ ID NOS:1-4. Traces for each probe using 5×10⁶ input target molecules are shown.All target concentrations were run in triplicate.

FIG. 12 shows real-time PCR amplification traces for probe SEQ ID NOS:11-14. Traces for each probe using 5×10⁶ input target molecules areshown. All target concentrations were run in triplicate.

FIG. 13 shows the absorbance spectrum of an oligonucleotide of SEQ IDNO: 15.

FIG. 14 shows the synthesis of a fluorescein aminooxy derivative.

FIG. 15 shows examples of aminooxy substituted reporter moieties.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a novel method of labeling oligonucleotides withreporter moieties during synthesis of the oligonucleotide. The methodpermits the attachment of several different reporter moieties to asingle oligonucleotide.

For the purposes of this invention, the term “reporter moiety” refers toa substituent that allows detection, either directly or indirectly, of acompound at low concentrations. Suitable reporter moieties include, butare not limited to, (1) enzymes, which produce a signal detectable, forexample, by colorimetry, fluorescence or luminescence, such ashorseradish peroxidase, alkaline phosphatase, beta-galactosidase orglucose-6-phosphate dehydrogenase; (2) chromophores, such asfluorescent, luminescent or dye compounds; (3) groups with an electrondensity which can be detected by electron microscopy or through theirelectrical property, such as by conductivity, amperometry, voltametry,or impedance measurements; and (4) groups which can be detected usingoptical methods, such as diffraction, surface plasma resonance orcontact angle variation, or physical methods, such as atomic forcespectroscopy, or the tunnel effect. Other suitable reporter moietiesinclude, but are not limited to, biotin, digoxigenin, peptides,proteins, antibodies, glycoproteins, and sugars.

In one embodiment, the method comprises forming an O-substituted oxime(“oxime”) bond between a reporter moiety having a nucleophile capable offorming an oxime bond with an oxo group (also referred to as anucleophile containing reporter moiety) and an oxo-substituted reactant.The oxime bond is completely orthogonal to reactions duringphosphoramidite oligonucleotide synthetic cycle and can be used as auniversal method for introduction of multiple modifications into anoligonucleotide. The oxime bond may be used to introduce almost anymodification into an oligonucleotide during synthesis or prior tosynthesis by modification of the solid support. The bond is unexpectedlystable, and remains intact during thermocycling. This method alsopermits the introduction of multiple different reporter moieties into anoligonucleotide.

The oxo-substituted reactant can be an oxo-substituted oligonucleotidewhich is linked to a solid support, an oxo-substituted nucleotide, anoxo-substituted nucleoside, an oxo-substituted nucleosidephosphoramidite, or a composition of Formula (II):

wherein R is H or alkyl, PG is a hydroxyl protecting group, such asthose commonly used in oligonucleotide synthesis, e.g. dimethoxytrityl(DMT), monomethoxytrityl (MMT), or trityl, and A is a linker used toattach an oligonucleotide to a solid support during synthesis of theoligonucleotide, such as the phosphate linkers, shown in 20a and 20b ofFIG. 3. Suitably, the alkyl is selected from a C₁₋₆ alkyl group, whichis substituted or unsubstituted, branched or unbranched, and saturatedor unsaturated. Suitable substituents include, but are not limited to,alkoxy, hydroxyl, cyano, amino, alkylamino, dialkylamino, halogen,alkylthio, and thiol. The oxo-substituted nucleotide and oxo-substitutednucleoside can be attached to a solid support.

The oxo-substituted oligonucleotides, oxo-substituted nucleotides,oxo-substituted nucleosides, and oxo-substituted nucleosidephosphoramidites for use in the present invention include thosecontaining the traditional nucleobases, such as adenine, guanine,cytosine, uracil and thymine, and those containing modified nucleobases.

The term “solid support” refers to any support that is compatible witholigonucleotide synthesis. For example, the following are suitable:glass, controlled pore glass, polymeric materials, polystyrene beads,coated glass, and the like.

In another embodiment, the method permits incorporation of anoxo-substituted nucleotide into an oligonucleotide followed by reactionwith a reporter moiety having a nucleophilic substituent capable offorming an oxime bond with the oxo group. The reporter moiety can beadded immediately after the oxo-substituted nucleotide is added to theoligonucleotide or the reporter moiety can be added after additionalnucleotides or oxo-substituted nucleotides have been added to theoligonucleotide. In another suitable embodiment, the novel methodpermits internal incorporation of a reporter moiety into anoligonucleotide as a reporter moiety substituted nucleotide which isincorporated into the oligonucleotide using standard phosphoramiditechemistry.

In another embodiment, the nucleophile containing reporter moiety can bereacted with an oxo-substituted reactant. The resulting composition, areporter moiety substituted reactant, is then used to derivatize a solidsupport, as in Example 3, and the derivatized support can serve as thefoundation for oligonucleotide synthesis by standard methods. AlthoughExample 3 demonstrates the attachment of an azo quencher compound tocontrolled pore glass, the method is more generally applicable to theattachment of a reporter moiety to any solid support that contains freereactive electrophile groups, including ketones and aldehydes. The solidsupport bound reporter moiety can be used conveniently in conjunctionwith automated oligonucleotide synthesizers to directly incorporate thereporter moiety into oligonucleotides during their synthesis.

The present method allows for multiple reporter moieties to beintroduced into a single oligonucleotide. The reporter moieties may bethe same or different. Use of different reporter moieties on a singleoligonucleotide allows detection of multiple signals using a singleoligonucleotide. Detection may be simultaneous or sequential.

The invention also provides novel azo compounds that are useful asfluorescence quenchers. The quenchers of this invention, which releaseenergy absorbed from fluorophores without emitting light, i.e. are “darkquenchers”, have the general formula shown below in Formula (I).

Each of R₁₋₆ is individually selected from the group consisting ofhydrogen, electron withdrawing groups such as halogens, NO₂, SO₃R_(S),SO₂N(R_(N))₂, CN, CNS, keto, and alkoxy groups, C₁-C₁₀ alkyl groups,aryl groups, and heteroaryl groups. R_(N) and R_(S) can be C₁-C₁₀ alkylgroups, which may be branched or unbranched and saturated orunsaturated, and substituted or unsubstituted, and aryl groups, whichmay be substituted or unsubstituted. Suitable substituents includeelectron withdrawing groups such as those described above.

R₇ can be any aryl group that can be joined to the conjugated ringsystem by an azo bond to form a compound that is capable of quenchingthe fluorescence of a fluorophore. Suitable aryl groups include phenyl,naphthyl, benzyl, xylyl, toluoyl, pyridyl, and anilinyl, among othergroups. R₇ can be substituted or derivatized with at least one linkinggroup for linking the quencher compound to other compounds of interest.

Y is a nucleophile-containing group capable of reacting with an oxogroup to form an oxime bond, such as aminooxy or hydrazine. In addition,any one of the R₁/R₂ pair, R₃/R₄ pair, R₄/R₅ pair and R₅/R₆ pair can becombined to form ring structures having five or six ring members. Thesering structures can be substituted with hydrogen, heteroatom-substitutedalkyl, halogen, alkenyl, alkoxy, alkoxy-alkyl, hydroxyl,trifluoromethyl, cyano, nitro, acyl, acyloxy, amino, alkylamino,dialkylamino, carboxyl, carbalkoxyl, carboxamido, mercapto, sulfamoyl,phenyl, and napthyl.

In addition, reactive substituents at R₁₋₆, such as amino, hydroxyl, andcarboxyl groups, can be attached to linking groups or other molecules ofinterest.

For purposes of this invention, the term “linking group” refers to achemical group that is capable of reacting with a “complementaryfunctionality” of a reagent, e.g., to the ketone group of aphosphoramidite, to form a bond that connects the azo quenching compoundof Formula (I) to the reagent. See R. 35 Haugland (1992) MolecularProbes Handbook of Fluorescent Probes and Research Chemicals, MolecularProbes, Inc., disclosing numerous modes for conjugating a variety ofdyes to a variety of compounds, which is incorporated herein byreference.

In one embodiment, R₇—Y is the compound of Formula (III) where the arylring is an anilinyl group which can be substituted with various groupsat positions L and L′.

L and L′ are independently selected from the group consisting ofsubstituted or unsubstituted C₁₋₁₀ alkyl and nucleophile-containingC₁₋₁₀ alkyl groups, wherein the C₁₋₁₀ alkyl groups are saturated orunsaturated. For example, in one embodiment, one of L or L′ can be anonreactive group (i.e., one that does not contain a nucleophile andcannot be modified to contain a nucleophile), such as an alkyl group,preferably an ethyl group, and the other can be a reactive group, suchas a hydroxyethyl group which can be modified further to a nucleophilicgroup such as aminooxy to facilitate linking the quencher to othermolecules of interest. One of skill in the art would recognize thathydroxy alkyl chains of any length could be used to modify the anilinylgroup.

A suitable embodiment of Formula (III) is shown in Formula (IV) below.

wherein Y is a nucleophile capable of reacting with an oxo group to forman oxime bond.

In one embodiment of Formula (I), the azo quencher compound has thestructure of Formula (V), wherein Y is an aminooxy group.

Suitable azo quencher precursor compounds have a primary amino group andhave the general structure of Formula (VI). Specific embodiments ofFormula (VI) include compounds 1 and 2.

The azo quenchers of Formula (I) are suitable for incorporation intooligonucleotides as is discussed above. The azo quenchers of Formula (I)can be linked to a variety of other useful compounds, provided thatsuitable reactive groups are present on those compounds. Such compoundsinclude antigens, antibodies, steroids, vitamins, drugs, haptens,metabolites, toxins, environmental pollutants, amino acids, proteins,carbohydrates, lipids, and the like.

Examples of other aminooxy substituted reporter moieties are shown inFIG. 15.

The invention also is directed to oligonucleotide compositionscontaining dye pairs, which include one of the disclosed quenchercompounds and a fluorophore that fluoresces on exposure to light of theappropriate wavelength. Suitable fluorophores in the dye pair are thosethat emit fluorescence that can be quenched by the quencher of the dyepair. In certain embodiments, the dye pair can be attached to a singlecompound, such as an oligonucleotide. In other embodiments, thefluorophore and the quencher can be on different compounds.

A wide variety of reactive fluorophores are known in the literature andcan be used with a corresponding quencher. Typically, the fluorophore isan aromatic or heteroaromatic compound and can be a pyrene, anthracene,naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole,benzothiazole, cyanine, carbocyanine, salicylate, anthranilate,coumarin, fluorescein, rhodamine or other like compound. Suitablefluorophores include xanthene dyes, such as fluorescein or rhodaminedyes, including 6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N;N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX). Suitable fluorophores also include the naphthylamine dyes thathave an amino group in the alpha or beta position. For example,naphthylamino compounds include 1-dimethylaminonaphthyl-5-sulfonate,1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalenesulfonate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Other fluorophores include coumarins, such as3-phenyl-7-isocyanatocoumarin; acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such asindodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5),indodicarbocyanine 5.5 (Cy5.5),3-(-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA);1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[2(or4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or TexasRed); BODIPY™ dyes; benzoxaazoles; stilbenes; pyrenes; and the like. Thefluorescent emission of certain fluorophores is provided below.

Fluorophore Emission Max 6-Carboxyfluorescein (6-FAM) 520 nmTetrachlorofluorescein (TET) 536 nm Hexachlorofluorescein (HEX) 556 nmCy3 570 nm Tetramethylrhodamine (TAMRA) 580 nm Cy3.5 596 nmCarboxy-x-rhodamine (ROX) 605 nm Texas Red 610 run Cy5 667 nm Cy5.5 694nm

The quencher of Formula (I) is capable of absorbing the fluorescentenergy in the range of about 500 to about 620 nm and therefore can beused to quench the fluorescence of fluorescein through Texas Red.

Many suitable forms of fluorophores are available and can be useddepending on the circumstances. With xanthene compounds, substituentscan be attached to xanthene rings for bonding with various reagents,such as for bonding to oligonucleotides. For fluorescein and rhodaminedyes, appropriate linking methodologies for attachment tooligonucleotides have also been described. See, for example, Khanna etal. U.S. Pat. No. 4,439,356, which is incorporated herein by reference;Marshall (1975) Histochemical J., 7:299-303, which is incorporatedherein by reference; Menchen et al., U.S. Pat. No. 5,188,934, which isincorporated herein by reference; Menchen et al., European PatentApplication No. 87310256.0, which is incorporated herein by reference;and Bergot et al., International Application PCT/U590/05565, which isincorporated herein by reference. Other quenchers could potentially beincorporated into an oligonucleotide using the method of the presentinvention. Some of these are shown in Table 1 below.

TABLE 1 Quencher Name/ODN λ_(max) modification Chemical structure (nm)Dabcyl

474 Eclipse (Disperse Red 13)

522 3′-BHQ-1

534 3′-BHQ-2

579 3′-BHQ-3

672 QSY7

560 QSY9

661 QSY21

661 QSY35

475

Suitably, when the dye pair is in a configuration in which fluorophoreis effectively quenched by the quencher dye, its fluorescence is reducedby at least a factor of 80%, and more preferably by 90%, 95%, or 98%,when compared to its fluorescence in the absence of quenching. Highlevels of quenching allow for the preparation of oligonucleotide probeshaving a high signal to noise ratio which is defined as the amount ofsignal present when the composition is in its maximal unquenched state(signal) versus its maximally quenched state (noise).

Probes having a high signal to noise ratio are desirable for thedevelopment of highly sensitive assays. To measure signal to noiseratios relative fluorescence is measured in a configuration where thequencher and fluorophore are within the Förster distance and thefluorophore is maximally quenched (background fluorescence or “noise”)and compared with the fluorescence measured when fluorophore andquencher are separated in the absence of quenching (“signal”). Thesignal to noise ratio of a dye pair of the invention will generally beat least about 2:1 but generally is higher. Signal to noise ratios aregenerally affected by the fluorophore-quencher pair, the quality of thesynthesis, and the oligonucleotide sequence.

Oligonucleotide probes that include a dye pair can be used to detecttarget oligonucleotides. In one method, the individual components of adye pair can be on opposing, hybridizable, self-complementary segmentsof a single oligonucleotide such that when the oligonucleotidehybridizes to itself in the absence of exogenous sequences, FRET occurs.The oligonucleotide probe is constructed in such a way that the internalhybridizing is disrupted and fluorescence can be observed when theoligonucleotide probe hybridizes to a complementary targetoligonucleotide. Such an oligonucleotide probe can be used to rapidlydetect target oligonucleotides having sequences that bind to theoligonucleotide probe. In another embodiment, a composition comprisestwo biomolecules, such as oligonucleotides, with a fluorophore attachedto one of the biomolecules and a quencher attached to the other.

Oligonucleotide probes lacking self-complementarity can also be utilizedin a similar manner. For example, a quencher and fluorophore can beplaced on an oligonucleotide that lacks the self-hybridizing propertysuch that the random-coil conformation of the oligonucleotide keeps thefluorophore and quencher within a suitable distance for fluorescencequenching. Such oligonucleotides can be designed so that when theyhybridize to desired target oligonucleotides the fluorophore andquencher are further apart and fluorescence can be observed.

Other DNA binding formats are also possible. For example, twooligonucleotide probes can be designed such that they can hybridizeadjacent to each other on a contiguous length of a targetoligonucleotide. The two probes can be designed such that when they arehybridized to the target oligonucleotide, a quencher on one of theoligonucleotide probes is within a sufficient proximity to a fluorophoreon the other oligonucleotide probe for FRET to occur. Binding of theoligonucleotide probes to the target oligonucleotide can be followed asa decrease in the fluorescence of the fluorophore.

Alternatively, a set of oligonucleotides that hybridize to each othercan be configured such that a quencher and a fluorophore are positionedwithin the Förster distance on opposing oligonucleotides. Incubation ofsuch an oligonucleotide duplex with another oligonucleotide thatcompetes for binding of one or both of the oligonucleotides would causea net separation of the oligonucleotide duplex leading to an increase inthe fluorescent signal of the fluorophore. To favor binding to thepolymer strands, one of the oligonucleotides could be longer ormismatches could be incorporated within the oligonucleotide duplex.

These assay formats can easily be extended to multi-reporter systemsthat have mixtures of oligonucleotides in which each oligonucleotide hasa fluorophore with a distinct spectrally resolvable emission spectrum.The binding of individual oligonucleotides can then be detected bydetermining the fluorescent wavelengths that are emitted from a sample.Such multi-reporter systems can be used to analyze multiplehybridization events in a single assay.

Oligonucleotides can also be configured with the disclosed quencherssuch that they can be used to monitor the progress of PCR reactionswithout manipulating the PCR reaction mixture (i.e., in a closed tubeformat). The assay utilizes an oligonucleotide that is labeled with afluorophore and a quencher in a configuration such that fluorescence issubstantially quenched. The oligonucleotide is designed to havesufficient complementarity to a region of the amplified oligonucleotideso that it will specifically hybridize to the amplified product. Thehybridized oligonucleotide is degraded by the exonuclease activity ofTaq polymerase in the subsequent round of DNA synthesis. Theoligonucleotide is designed such that as the oligomer is degraded, oneof the members of the dye pair is released and fluorescence from thefluorophore can be observed. An increase in fluorescence intensity ofthe sample indicates the accumulation of amplified product.

Ribonucleic acid polymers can also be configured with fluorophores andquenchers and used to detect RNase. For example, a dye pair can bepositioned on opposite sides of an RNase cleavage site in an RNasesubstrate such that the fluorescence of the fluorophore is quenched.Suitable substrates include oligonucleotides that have a single-strandedregion that can be cleaved and that have at least one internucleotidelinkage immediately 3′ to an adenosine residue, at least oneinternucleotide linkage immediately 3′ to a cytosine residue, at leastone internucleotide linkage immediately 3′ to a guanosine residue and atleast one internucleotide linkage next to a uridine residue andoptionally can lack a deoxyribonuclease-cleavable internucleotidelinkage. To conduct the assay, the substrate can be incubated with atest sample for a time sufficient for cleavage of the substrate by aribonuclease enzyme, if present in the sample. The substrate can be asingle-stranded oligonucleotide containing at least one ribonucleotideresidue at an internal position. Upon cleavage of the internalribonucleotide residue, the fluorescence of the fluorophore, whoseemission was quenched by the quencher, becomes detectable. Theappearance of fluorescence indicates that a ribonuclease cleavage eventhas occurred, and, therefore, the sample contains ribonuclease activity.This test can be adapted to quantitate the level of ribonucleaseactivity by incubating the substrate with control samples containingknown amounts of ribonuclease, measuring the signal that is obtainedafter a suitable length of time, and comparing the signals with thesignal obtained in the test sample.

The invention also provides kits that comprise a labeled oligonucleotideor an azo quencher of the present invention. The kit can also containinstructions for use. Such kits can be useful for practicing thedescribed methods or to provide materials for synthesis of thecompositions as described. Additional components can be included in thekit depending on the needs of a particular method. For example, wherethe kit is directed to measuring the progress of PCR reactions, it caninclude a DNA polymerase. Where a kit is intended for the practice ofthe RNase detection assays, RNase-free water could be included. Kits canalso contain negative and/or positive controls and buffers.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope. In particularthe following examples demonstrate synthetic methods for obtaining thecompounds of the invention. Starting materials useful for preparing thecompounds of the invention and intermediates thereof, are commerciallyavailable or can be prepared from commercially available materials usingknown synthetic methods and reagents. All oligonucleotide sequences arewritten from the 5′-terminus on the left to the 3′-terminus on theright.

Example 1 Synthesis of aminooxy activated(1-nitro-4-naphthylazo)-N,N-diethanolaniline quencher (6)

Synthesis was performed as shown in Scheme 1 in FIG. 1. To the solutionof 0.36 g (0.1 mmol) alcohol (4), 0.17 g (0.1 mmol)N-hydroxy-phthalimide, and 0.27 g (0.1 mmol) of triphenylphosphine in 10mL of THF was added 0.18 mL (0.1 mmol) of diethylazodicarboxylate(DEAD). After overnight stirring the reaction mixture was concentratedunder diminished pressure. Flash chromatography with 1:4 EtOAc/hexanesprovided 150 mg of (5). TLC: R_(f) 0.75 (EtOAc/hexanes—60/40). ¹H NMR(CDCl₃) δ 9.04 (d, J=8.4 Hz, 1H), 8.68 (d, J=8.4 Hz, 1H), 8.34 (d, J=8.4Hz, 1H), 8.03 (d, J=8 Hz, 2H), 7.7-7.9 (m, 7H), 6.85 (d, J=8 Hz, 2H),4.46 (t, J=7.5 Hz, 2H), 3.92 (t, J=7.5 Hz 2H), 3.72 (q, J=8 Hz, 2H),1.34 (t, J=8 Hz 3H).

The solution of 10 mg (5) in 2 mL of concentrated ammonia solution inethanol was incubated overnight at 55° C. The solvent was removed underdiminished pressure to provide 6) that was used further withoutpurification.

Example 2 Synthesis of Ketone Phosphoramidite (16)

Synthesis was performed as shown in Scheme 2 of FIG. 2.

N-Fmoc-3-aminopropyl solketal (10): 3-Aminopropyl solketal (9) wassynthesized starting from commercially available solketal (7) accordingto the procedure of Misiura et al (Misiura, K., Durrant, I., Evans, M.R., Gait, M. J. (1990) Nucleic Acids Research, v. 18, No. 15, pp.4345-4354, which is incorporated herein by reference). (9) was usedcrude without vacuum distillation for the next step. The crude product(9) (12.85 g; 68 mmol) was dissolved in dry CH₃CN (100 mL) withstirring. NaHCO₃ (4.2 g; 50 mmol) was added followed by Fmoc-OSu (16.9g; 50 mmol). The reaction mixture was stirred at room temperatureovernight. The solvent was evaporated and the oily residue waspartitioned between EtOAc (500 mL) and 5% NaHCO₃ (150 mL). The organiclayer was separated and washed with 5% NaHCO₃ (2×150 mL), brine (150mL), and dried over anhydrous. Na₂SO₄. The product (10) was isolated byflash chromatography on a silica gel column (5×20 cm) loading fromEtOAc:CH₂Cl₂:petroleum ether (PE) (15:15:70) and eluting withEtOAc:CH₂Cl₂:PE (1:1:2). The isolated product (10) had R_(f) of 0.4 byTLC in EtOAc:CH₂Cl₂:PE (1:1:1). Yield: 20.95 g of oil. ¹H NMR (CDCl₃) δ1.35 (s, 3H), 1.45 (s, 3H), 1.81 (m, 2H), 3.34 (q, 2H), 3.47-3.60 (m,4H), 3.75 (dd, 1H), 4.07 (dd, 1H), 4.22-4.32 (m, 2H), 4.42 (d, 2H), 5.29(br. t, 1H), 7.33 (dt, 2H), 7.42 (t, 2H), 7.62 (d, 2H), 7.78 (d, 2H).

1-O—(N-Fmoc-3-aminopropyl)glycerol (11): Crude compound (10) (5 g; 12.1mmol) was dissolved in THF (15 mL) and treated with 2M HCl (5 mL). Theresulting emulsion was shaken at room temperature with occasionalsonication until it became homogeneous. It was then left at roomtemperature for additional hour. The reaction mixture was concentratedin vacuum, and the resulting oil was co-evaporated with absolute EtOH(3×20 mL). The reaction product (R_(f) of ˜0.3 in EtOAc:CH₂Cl₂:MeOH(10:10:1)) was isolated by silica gel chromatography (5×20 cm) using agradient 0-5% MeOH in EtOAc:CH₂Cl₂ (1:1). Fractions containing pureproduct were pooled and concentrated to give oily residue, whichcrystallized upon vacuum drying. Yield: 2.64 g of a white solid (11). ¹HNMR (DMSO-d6) δ 1.63 (m, 2H), 3.05 (q, 2H), 3.25-3.41 (m, 6H), 3.53-3.60(m, 1H), 4.21 (t, 1H), 4.30 (d, 2H), 4.47 (t, 1H), 4.60 (d, 1H), 7.27(t, 1H), 7.33 (dt, 2H), 7.42 (t, 2H), 7.69 (d, 2H), 7.89 (d, 2H).

1-O-DMT-3-O—(N-Fmoc-3-aminopropyl)glycerol (12):1-O—(N-Fmoc-3-aminopropyl)glycerol (11) (2.64 g; 7.1 mmol) was dissolvedin dry pyridine (50 mL) and treated with DMT-C1 (2.65 g; 7.8 mmol). Thereaction mixture was stirred at room temperature overnight and quenchedwith MeOH (5 mL). It was then concentrated to oil under reducedpressure. The residue was dissolved in EtOAc (˜300 mL) and extractedwith saturated NaHCO₃ (3×100 mL) followed by brine (100 mL). The organicphase was separated, dried over anhydrous Na₂SO₄ and concentrated tooil. The product (12) was isolated by silica gel chromatography using agradient 33-66% EtOAc in petroleum ether (“PE”). Yield: 4.03 g (84%) ofwhite foam (12). TLC showed one spot at R_(f) ˜0.6 in EtOAc:PE (2:1). ¹HNMR (CDCl₃) δ 1.68-1.80 (m, 2H), 2.57 (br d, 1H), 3.17-3.34 (m, 4H),3.43-3.61 (m, 4H), 3.79 (s, 6H), 3.93-4.00 (m, 1H), 4.22 (t, 1H), 4.41(d, 2H), 5.20 (br t, 1H), 6.82-6.86 (m, 4H), 7.21-7.46 (m, 13H), 7.61(d, 2H), 7.77 (d, 2H).

1-O-DMT-3-O-(3-aminopropyl)glycerol (13): Compound (12) (3.82 g; 5.67mmol) was dissolved in i-PrOH (100 mL) and sodium borohydride (4 g) wasadded in portions with stirring. The suspension was heated at 70° C. for2 hours. TLC analysis in EtOAc:TEA (99:1) revealed the disappearance ofthe starting material (R_(f) ˜0.75) and formation of deprotected productat the start. The reaction was carefully quenched with 10% sodiumhydroxide (32 mL), transferred into a reparatory funnel and partitionedwith 300 mL of ethyl acetate. The organic phase was separated, washedwith saturated NaHCO₃ (3×100 mL) followed by brine (100 mL), and driedover sodium sulfate. It was then concentrated in vacuum to give oilyresidue, which was co-evaporated with dry acetonitrile (50 mL). Thiscrude material (13) was used in the next step without furtherpurification.

Pentafluorophenyl 5-oxohexanoate (14): 5-Oxohexanoic acid (2.6 g; 20mmol) was dissolved in CH₂Cl₂ (50 mL). N,N-Diisopropylethylamine (10.4mL, 60 mmol) was added followed by pentafluorophenyl trifluoroacetate(3.61 mL; 21 mmol). The reaction mixture was kept at room temperaturefor 1 hour and evaporated. The residue was resuspended in EtOAc:Hexanes(1:1) and loaded on a silica gel column (5×20 cm) equilibrated anddeveloped with the same mixture. Fractions containing the product (14)(R_(f) ˜0.7) were pooled and concentrated to give 4.7 g (79%) ofyellowish oil after drying in vacuum. ¹H NMR (CDCl₃) δ 2.05 (m, 2H),2.18 (s, 3H), 2.61 (t, 2H), 2.74 (t, 2H).

1-O-DMT-3-O—(N-(5-oxohexanoyl)-3-aminopropyl)glycerol (15): The crudeproduct (13) was dissolved in dry CH₃CN (50 mL) and treated withN,N-diisopropylethylamine (2.6 mL, 15 mmol) and (14) (1.68 g, 5.67mmol). The mixture was allowed to react at room temperature for 2 hours.The reaction mixture was evaporated in vacuum and the residue wasreconstituted in EtOAc (50 mL). The product was isolated by silica gelchromatography (4×25 cm) loading from 1% triethylamine (TEA) in EtOAcand eluting with MeOH:EtOAc:TEA (5:95:1). Fractions containing a singlecomponent (R_(f) 0.35) were pooled and concentrated in vacuum to yieldthe title compound (15) (2.70 g, 85%) as slightly orange oil. ¹H NMR(DMSO-d6) δ 1.60 (m, 2H), 1.66 (m, 2H), 2.03 (t, 2H), 2.05 (s, 3H), 2.40(t, 2H), 2.94 (d, 2H), 3.04 (q, 2H), 3.35-3.46 (m, 4H), 3.72-3.79 (m,7H; OCH₃ singlet at 3.74), 4.84 (d, 1H), 6.88 (d, 4H), 7.19-7.42 (m,9H), 7.72 (t, 1H).

1-O-DMT-3-O—(N-(5-oxohexanoyl)-3-aminopropyl)glycerol2-O—(N,N-diisopropyl-(2-cyanoethyl)phosphoramidite) (16): Alcohol (15)(1.35 g, 2.4 mmol) and diisopropylammonium tetrazolide (206 mg, 1.2mmol) were dissolved in anhydrous CH₃CN (30 mL) under argon atmosphere.2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphoramidite (0.953 mL, 3.0mmol) was added with stirring at room temperature, and the reactionmixture was stirred overnight. The solvent was evaporated, the residuewas reconstituted in EtOAc (200 mL) and washed with saturated NaHCO₃(3×50 mL) followed with brine (50 mL). The organic layer was dried overanhydrous Na₂SO₄ and the solvent evaporated under reduced pressure. Theoily residue was purified by silica gel chromatography eluting withEtOAc:TEA (95:5). Fractions containing pure product (16), which moves asa double spot on TLC (R_(f) ˜0.55; EtOAc:TEA (95:5)), were pooled andconcentrated in vacuum to give 1.74 g of colorless oil (16). ¹H NMR(DMSO-d6) δ 1.01-1.17 (m, 12H), 1.56 (m, 2H), 1.66 (m, 2H), 2.02 (m,2H), 2.05 (s, 3H), 2.39 (m, 2H), 2.65 (t, 1H), 2.77 (t, 1H), 2.97-3.16(m, 4H), 3.36-3.81 (m, 15H; OCH₃ singlets at 3.73 and 3.74), 6.88 (m,4H), 7.19-7.44 (m, 9H), 7.69 (t, 1H). ³¹P NMR (DMSO-d6) δ 148.19 and148.64.

Example 3 Synthesis of aminooxy conjugated CPG supports with(1-nitro-4-naphthylazo)-N,N-diethanolaniline quencher (20a and 20b)

Synthesis was performed as shown in Scheme 3 in FIG. 3.

Synthesis of ketone substituted controlled pore glass (CPG) supports:Spacer C3 CPG (2 g; 44 μmol/g) was placed in a 50 mL peptide synthesisreactor and detritylated by treating with several portions (30 mL) of 3%dichloroacetic acid in dichloromethane (until all the color was washedoff the support). It was then washed with CH₃CN (5×50 mL; last two timeswith anhydrous CH₃CN) and “activator” (30 mL; 0.45 M5-ethylthio-1H-tetrazole in anhydrous. CH₃CN) under argon atmosphere.The CPG (18) was then treated with a solution of appropriatephosphoramidite (phosphoramidite (17a) which was synthesized accordingto published procedure: Dey, S. Shepard, T. (2001) Org Lett, v. 3, pp.3983-3986) which is incorporated herein by reference; (250 μmol) in 10mL of anhydrous CH₃CN mixed with 10 mL of “activator” at roomtemperature for 30 minutes under Ar purge. The reagents were removed byvacuum suction and replaced with a fresh portion. The coupling reactionwas repeated; modified CPG was filtered off and washed with CH₃CN (5×30mL). The solid support was treated with 0.1 M I₂ in THF/Py/H₂O (3×30 mL;5 minutes each treatment), and washed with CH₃CN (5×30 mL). Unreactedhydroxyls were capped by treating with Ac₂O:MeIm:Py (10:10:80) (3×30 mL;5 minutes each treatment). The derivatized CPG (19a) was washed withCH₃CN (5×30 mL), CH₂Cl₂ (3×30 mL), and dried in vacuum overnight.DMT-loading was usually above 30 μmol/g.

Attachment of the quencher to ketone substituted support: To thesolution of 10 mg quencher (6) was added 0.1 g ketone substitutedsupport (19a) and incubated overnight at room temperature. The resultingsupport (20a) was filtered and washed with three 1 ml portions ofacetonitrile and then used in oligonucleotide synthesis.

Example 4 Quenching Efficiency of Amino-Oxy Quencher Derivatives

This example demonstrates the signal to noise ratio (S:N) ratio ofoligonucleotides containing both fluorescein and the azo quencher asprepared in Examples 1 through 3. Fluorescence-quenched probes areemployed in a variety of applications in molecular biology. One methodto assess if a given fluorophore and a quencher function well togetheris by measurement of a signal to noise ratio (S:N), where relativefluorescence is measured in the native configuration (backgroundfluorescence or “noise”) and compared with fluorescence measured whenfluorophore and quencher are separated (“signal”).

Oligonucleotide Synthesis. The following oligonucleotides weresynthesized using standard phosphoramidite chemistry and the aminooxyquencher reagents described in Example 3, supra. Oligonucleotides werepurified by HPLC. Dual-labeled oligonucleotides were made with the novelaminooxy (1-nitro-4-naphthylazo)-N,N-diethanolaniline quencher (6) ofthe invention at the 3′-end of the probe with the fluorescein reportergroup placed at the 5′ end (6-FAM, single isomer 6-carboxyfluorescein,Glen Research, Sterling, Va.). The same sequence was made usingdifferent methods of quencher attachment, including conjugation of theaminooxy quencher (6) post-synthetically (IBAOket), conjugation of theaminooxy quencher (6)-dU-CPG at the time of synthesis (IBAOdU, FormulaV, supra), and conjugation of the aminooxy quencher (6)-ketone-CPG atthe time of synthesis (IABAOC7, Formula (IV), supra). For comparisonpurposes, an oligonucleotide was made that incorporates a commerciallyavailable quenching group, Eclipse Quencher™-CPG (Epoch Biosciences,Bothell, Wash.). The Eclipse Quencher probe does not contain an aminooxynucleophile. In order to make 3′- or internal FAM modification usingketone phosphoramidite (SEQ ID NO: 5), FAM-oxime conjugate has to beacetylated with acetic anhydride capping reagent prior followingphosphoramidite cycle.

Electrospray-ionization liquid chromatography mass spectroscopy(ESI-LCMS) of each oligonucleotide probe was performed using an OligoHTCS system (Novatia, Princeton, N.J.), which consisted ofThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processingsoftware and Paradigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.).Protocols recommended by manufacturers were followed. Experimental molarmasses for all compounds were within 0.02% of expected molar mass,confirming the identity of the compounds synthesized.

TABLE 2 Probe Sequence SEQ ID NO: 1 FAM-CCAGCGACCCTGATTATGGCCTCCCT-IBAOket SEQ ID NO: 2 FAM-CCAGCGACCCTGATTATGGCCTCCCT-IBAOdU SEQ ID NO: 3FAM-CCAGCGACCCTGATTATGGCCTCCCT-IBAOC7 SEQ ID NO: 4FAM-CCAGCGACCCTGATTATGGCCTCCCT- Eclipse

As representative of the final structure obtained using reagentsoutlined in Example 3 in oligonucleotide synthesis, the chemical linkagebetween aminooxy quencher and the 3′-end of oligonucleotide SEQ ID NO: 3is shown below (Formula (VII)).

Signal to Noise (S:N) Assay of Fluorescence-Quenched Linear Probes:Oligonucleotides were evaluated for quenching efficiency in a pre- andpost-nuclease degradation assay. Probe oligonucleotides (SEQ ID NOS:1-4) were individually resuspended at 100 nM concentration in HPLC-gradewater. From this stock solution, 2 ml of 100 nM probe solution wasprepared with STNR Buffer, comprising 10 mM Tris pH 8.3, 50 mM KCl, 5 mMMgCl₂, 1 mM CaCl₂, which was split into two identical 1 mL fractions.One fraction was retained without enzyme treatment as backgroundcontrol. The second fraction was subjected to nuclease degradation asfollows. Micrococcal nuclease, 15 units (Roche, 15 U/ul), was added tothe oligonucleotide solution and incubated at 37° C. for 1 hour.Relative fluorescence intensity for each sample was measured with a PTIQuantaMaster Model C-60 cuvette-based spectrofluorometer (PhotonTechnology International, Monmouth Jct., NJ). The fluorescencemeasurement of the solution containing intact probe constituted the“background” or “noise” component of the assay. The fluorescencemeasurement of the solution containing degraded probe (nuclease treated)constituted the “signal” component of the assay. Signal to noise ratios(S:N) were calculated.

TABLE 3 Signal to Noise ratios for Fluorescence- Quenched LinearOligonucleotides. Dye/Quencher RFU RFU Probe ID 5′-3′ Background SignalS:N Ratio SEQ ID NO: 1 FAM-IBAOket 9.35E+05 7.25E+06 8 SEQ ID NO: 2FAM-IBAOdU 6.32E+05 9.13E+06 14 SEQ ID NO: 3 FAM-IBAOC7 4.85E+058.06E+06 17 SEQ ID NO: 4 FAM-Eclipse 8.99E+05 1.19E+07 15 RFU = relativefluorescence units

As shown in Table 3, the novel aminooxy attached quenchers (6) arecapable of quenching a fluorescein with similar efficiency as a commonlyemployed commercially available quencher group.

Example 5 Use of Aminooxy-Quenchers in Fluorescent Probes in aQuantitative Real-Time PCR Assay

Fluorescence-quenched probes can be employed to detect a target nucleicacid sequence. Commonly, such detection is linked to an amplificationstep, such as the polymerase chain reaction (PCR). This exampledemonstrates that use of fluorescent probes modified with aminooxyquenchers function in a relevant application, a real-time PCR assay.

Oligonucleotide primers were synthesized using standard phosphoramiditechemistry, desalted, and employed without additional purification. Probeoligonucleotides employed are the same compounds studied in Example 4supra, SEQ ID NOS: 1-4. Primers employed are:

Forward Primer: HP48 For AGAAGGTCATCATCTGCCATCG SEQ ID NO: 5 ReversePrimer: HP48 Rev TCCAGACTTTGGCTGTTCGGAT SEQ ID NO: 6

The target nucleic acid is SEQ ID NO: 7, a 150 base pair (bp) ampliconderived from the human bHLH protein PTF1A gene (Genbank #NM_(—)178161),cloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, Calif.), and ishereafter referred to as the “p48-gene target”.

Target Nucleic Acid Sequence:

SEQ ID NO: 7 HP48 For                                  HP48 ProbeAGAAGGTCATCATCTGCCATCGGGGCACCCGGTCCCCCTCCCCCAGCGACCCTGATTATGGCCTCCCTCCCCTAGCAGGACACTCTCTCTCATGGACTGATGAAAAACAACTCAAGGAACAAAATATTATCCGAACAGCCAAAGTCTGGA       HP48 Rev

PCR amplification was done using the thermostable DNA polymeraseImmolase™ (Bioline, Randolph, Mass.), 800 uM dNTPs, and 3 mM MgCl₂.Reactions were carried out in a 25 μL volume and comprised 200 nM eachof the amplification primers and fluorescent quenched probe, 500, 50,000and 5,000,000 copies of target DNA. Cycling conditions were 50° C. for 2min, 95° C. for 10 min, then 40 cycles of 2-step PCR with 95° C. for 15sec and 60° C. for 1 min. PCR and fluorescence measurements were doneusing an ABI Prism™ 7700 Sequence Detector (Applied Biosystems Inc.,Foster City, Calif.). All data points were performed in triplicate. Thecycle threshold (Ct) value is defined as the cycle at which astatistically significant increase in fluorescence is detected abovebackground. A lower Ct value is indicative of a higher concentration oftarget DNA. The assays were performed using an identical amount of inputtarget DNA (5×10²-5×10⁴-5×10⁶ copies of the PTF1a p48-gene targetplasmid). Relative fluorescence levels collected during PCR for eachprobe were graphically plotted against cycle number and are shown inFIGS. 1-5.

The multicomponent view of a 40-cycle real-time PCR reaction using probeSEQ ID NO: 1, which incorporates the aminooxy(1-nitro-4-naphthylazo)-N,N-diethanolaniline quencher (6) attachedpost-synthetically (IBAOket), is shown in FIG. 6. The fluorescencebaseline remained flat until cycle 18, when product first reacheddetectable levels as a result of PCR amplification. The oxime bond wasstable in the employed reaction conditions and no elevation of baselinebackground fluorescence was observed. The oxime bond is thereforesuitable for use in these reaction conditions, which are similar toconditions commonly employed in many molecular biology applications.

Amplification traces for probe SEQ ID NO: 1, which incorporates theaminooxy (1-nitro-4-naphthylazo)-N,N-diethanolaniline quencher (6)attached post-synthetically (IBAOket), are shown in FIG. 6. The resultsshowed good clustering of triplicate reactions and clearly distinguishedbetween different input concentrations of the target nucleic acid.

Amplification traces for probe SEQ ID NO: 2, which incorporates theaminooxy (1-nitro-4-naphthylazo)-N,N-diethanolaniline quencher (6)attached during synthesis as a CPG conjugate via a dU base linkage(IBAOdU), is shown in FIG. 7. The results showed good clustering oftriplicate reactions and clearly distinguished between different inputconcentrations of the target nucleic acid.

Amplification traces for probe SEQ ID NO: 3, which incorporates theaminooxy (1-nitro-4-naphthylazo)-N,N-diethanolaniline quencher attachedduring synthesis as a CPG conjugate via direct linkage to the 3′-end ofthe oligonucleotide (IBAOC7), are shown in FIG. 8. The results showedgood clustering of triplicate reactions and clearly distinguishedbetween different input concentrations of the target nucleic acid.

Amplification traces for probe SEQ ID NO: 4, which incorporatescommercial Eclipse Quencher attached during synthesis as a CPG conjugatevia direct linkage to the 3′-end of the oligonucleotide (Eclipse), areshown in FIG. 9. The results showed good clustering of triplicatereactions and clearly distinguished between different inputconcentrations of the target nucleic acid.

The real-time PCR results for all 4 probes were plotted together for asingle target concentration, 5×10⁶ and are shown in FIG. 10. Theabsolute change in fluorescence (ΔRf) varied between probes. Thistypically results from variable quality of purification at the time ofsynthesis. Actual sensitivity to quantitative detection of the inputtarget nucleic acid was nearly identical between probes and quenchersemployed.

Table 4 summarizes the real-time PCR results and demonstrates that alloligonucleotides provided similar Ct values regardless of method ofquencher attachment and functioned with similar performance in thisapplication.

TABLE 4 Relative Ct Values for Probes SEQ ID NOS: 1-4 in Real Time PCRAssay. Ave. Ct Ave. Ct Ave. Ct Probe Target 5 × 10⁶ Target 5 × 10⁴Target 5 × 10² SEQ ID NO: 1 17.9 25.3 31.4 SEQ ID NO: 2 17.1 24.5 30.5SEQ ID NO: 3 17.2 24.3 30.8 SEQ ID NO: 4 16.9 24.0 30.1

As shown in Table 4, probe compositions comprising the new aminooxy(1-nitro-4-naphthylazo)-N,N-diethanolaniline quencher (6) of theinvention performed well in a quantitative real-time PCR assay and werefunctionally interchangeable with probes that contain other quenchermoieties.

Example 6 Use of Internal Aminooxy-Du Quenchers in Fluorescent ProbesFunction in a Quantitative Real-Time PCR Assay

Quencher groups are commonly placed at the end of a probe sequence forease of synthesis. The new aminooxy quencher permits internalincorporation of quencher as a base modified aminooxy quencher-dUmoiety. This example demonstrates that use of fluorescent probesmodified with internal aminooxy-quenchers function better in a real-timePCR assay than standard end-quenched probes.

Dual-labeled oligonucleotides with internal modifications (SEQ ID NOS:12-14) were made using ketone-dU phosphoramidite (synthesized accordingto published procedure: Dey & Shepard, (2001) Org. Lett., v. 3, pp.3983-3986, which is incorporated by reference herein) followed byinterconjugation with 300 μL of 10 mM solution (per 1 μmole of theoligonucleotide on the solid support) of the aminooxy-quencher reagent(6) in ethanol at the time of synthesis. After 2 hours, excessaminooxy-quencher was removed, the solid support was washed with 1 mL ofacetonitrile and the oligonucleotide was extended using standardphosphoramidite chemistry.

Oligonucleotide primers were synthesized using standard phosphoramiditechemistry, desalted, and were used in the assay without additionalpurification. Primer and probe oligonucleotides employed are shownbelow. Probes with internal quencher modifications had a C3 spacer groupplaced at the 3′-end in place of the quencher group to block extensionduring PCR. Oligonucleotides were synthesized as described above.

TABLE 5 Sequence SEQ ID NO: 5′ FAM-ATGGCGGTTCTCATGCTGGCAAC-IBAOC7 3′ SEQID NO: 11 5′ FAM-ATGGCGGT(iIBAOdU)CTCATGCTGGCAAC-C3sp 3′ SEQ ID NO: 125′ FAM-ATGGCGGTT(iIBAOdU)TCATGCTGGCAAC-C3sp 3′ SEQ ID NO: 135′ FAM-ATGGCGGTTCTC(iIBAOdU)TGCTGGCAAC-C3sp 3′ SEQ ID NO: 145′ AACTCTGAAGTCATCCTGCCAGTC 3′ SEQ ID NO: 8 5′ CTTCAGGTTGTGGTAAACCTCTGC3′ SEQ ID NO: 9

The forward and reverse primers are shown in SEQ ID NOS: 8 and 9.Internal aminooxy-quencher-dU is notated by (iIBAOdU). SEQ ID NO: 11represents a traditional probe with 3′-terminal quencher placement. SEQID NO: 12 has an internal aminooxy-quencher-dU substitution for aninternal dT base at position 9 from the 5′-end. SEQ ID NO: 13 has aninternal aminooxy-quencher-dU substitution for an internal dC base atposition 10 from the 5′-end, which results in a favorable U:G basepairing event upon hybridization. SEQ ID NO: 14 has an internalaminooxy-quencher-dU substitution for an internal dA base at position 13from the 5′-end, which results in an unfavorable U:T base pairing eventupon hybridization. The aminooxy-quencher-dU base is compound (20a_.

The target nucleic acid is SEQ ID NO: 10, a 162 base pair (bp) ampliconderived from the human Enolase gene (Genbank #NM_(—)001428), cloned intothe pCRII-TOPO vector (Invitrogen, Carlsbad, Calif.), and is hereafterreferred to as the “hEnolase-gene target”.

Target Nucleic Acid Sequence:

SEQ ID NO: 10 Enolase For                                Enolase ProbeAACTCTGAAGTCATCCTGCCAGTCCCGGCGTTCAATGTCATCAATGGCGGTTCTCATGCTGGCAACAAGCTGGCCATGCAGGAGTTCATGATCCTCCCAGTCGGTGCAGCAAACTTCAGGGAAGCCATGCGCATTGGAGCAGAGGTTTACCACAACCTGAAG     Enolase Rev

PCR amplification was performed using the thermostable DNA polymeraseImmolase™ (Bioline, Randolph, Mass.), 800 uM dNTPs, and 3 mM MgCl₂.Reactions were carried out in a 25 μL, volume and comprised 200 nM eachof the amplification primers and fluorescent quenched probe, 500, 50,000and 5,000,000 copies of target DNA. Cycling conditions were 50° C. for 2min, 95° C. for 10 min, then 40 cycles of 2-step PCR with 95° C. for 15sec and 65° C. for 1 min. PCR and fluorescence measurements were doneusing an ABI Prism™ 7700 Sequence Detector (Applied Biosystems Inc.,Foster City, Calif.). All data points were performed in triplicate. Thecycle threshold (Ct) value is defined as the cycle at which astatistically significant increase in fluorescence is detected abovebackground. A lower Ct value is indicative of a higher concentration oftarget DNA. The assays were performed using an identical amount of inputtarget DNA (5×10²-5×10⁴-5×10⁶ copies of the hEnolase-gene targetplasmid). Relative fluorescence levels collected during PCR for eachprobe were graphically plotted against cycle number. The real-time PCRresults for all 4 probes are plotted together for a single targetconcentration, 5×10⁶ and are shown in FIG. 12. The absolute change influorescence (ΔRf) varied noticeably between probes. In this case,probes had similar quality and the differences in fluorescence relatesto different potency of quenching that varies with quencher placement.Actual sensitivity to quantitative detection of the input target nucleicacid varied between probes and is quantified in Table 6 below.

TABLE 6 Relative Ct Values for Probes SEQ ID NOS: 10-13 in Real Time PCRAssay. Ave. Ct Ave. Ct Ave. Ct Probe Target 5 × 10⁶ Target 5 × 10⁴Target 5 × 10² SEQ ID NO: 11 19.8 26.5 33.3 SEQ ID NO: 12 18.6 25.4 32.2SEQ ID NO: 13 19.1 25.8 32.6 SEQ ID NO: 14 19.1 25.7 32.6

Probe compositions comprising the new aminooxy(1-nitro-4-naphthylazo)-N,N-diethanolaniline quencher (6) placedinternally on a dU base show superior properties in a real-time PCRassay compared with standard 3′-quencher probes. Detection limits wereimproved by ˜1 Ct value, which corresponds to about double detectionsensitivity.

Example 7 Absorbance Spectrum

This example shows an absorbance spectrum of an oligonucleotide modifiedat its 5′terminus to contain the azoquencher (6). The oligonucleotidewas made using standard automated phosphoramidite nucleotide syntheticmethods where the last addition cycle was carried out with the molecule(6). The composition of the oligonucleotide is shown below.

SEQ ID NO: 15 (Azo-Quencher)-CAGAGTACCTGA

Once synthesized, the oligonucleotide was suspended in HPLC-grade waterat 400 nM concentration. Optical absorbance was measured in 10 mM TrispH 8.0, 1 mM EDTA (TE buffer) with a sub-micro quartz cuvette with 1-cmpath length in a Hewlett Packard Model 8453 spectrophotometer (HewlettPackard, Palo Alto, Calif.). Absorbance density was recorded from 220 nmto 700 nm and is shown in FIG. 13.

As shown in FIG. 13, the absorbance spectrum was broad, ranging from 420to 620 nm, with peak absorbance at 531 nm. This absorbance rangeoverlaps with the fluorescence emission of a wide variety offluorophores commonly used in molecular biology applications. For FRETbased quenching mechanisms, this spectrum is positioned to offer maximumquenching capacity for dyes in the spectral range of fluorescein.

Example 8

The aminooxy group is introduced to a reporter moiety via the Mitsunobureaction between alcohol (21) and N-hydroxyphthalimide followed byphthalimide hydrolysis. (Scheme 4 in FIG. 4).

This method can be used for derivatization of fluorophores, quenchers,biotin, peptides and other reporter moieties stable to basic conditions.

Example 9

In case of base labile molecules, such as some peptides, proteins,reporter moieties having alkylamino function, the aminooxy group isintroduced by reaction with corresponding NHS ester (28), followed byremoval of acid liable MMT group. (Scheme 5 in FIG. 5).

Example 10 Synthesis of Fluorescein Aminooxy Derivative (33)

This example demonstrates the chemical synthesis of the compound offormula:

The synthesis was as shown in FIG. 14 below. To the solution of 0.72 g(0.14 mmol) alcohol (31), 0.23 g (0.14 mmol) N-hydroxy-phthalimide, and0.36 g (0.14 mmol) of triphenylphosphine in 10 mL of THF was added 0.26mL (0.15 mmol) of DEAD. After overnight stirring the reaction mixturewas concentrated under diminished pressure. Flash chromatography with1:4 EtOAc/hexanes provided 380 mg of (32). TLC: R_(f) 0.55(EtOAc/hexanes—60/40). ¹H NMR (CDCl₃) δ 8.13 (d, J=8.4 Hz, 1H), 8.07 (d,J=8.4 Hz, 1H), 7.7-7.9 (m, 4H), 7.49 (s, 1H), 7.03 (d, 2.4 Hz, 2H),6.75-6.83 (m, 4H), 6.42 (t, J=6.5 Hz, 1H), 4.21 (t, J=6.5 Hz, 2H) 3.45(dd, J=8.5, J=6.5, 2H), 1.35-1.80 (m, 8H), 1.36 (s, 18H).

The solution of 10 mg (32) in 2 mL of concentrated ammonia solution inethanol was incubated overnight at 55° C. The solvent was removed underdiminished pressure to provide (33) that was used further withoutpurification.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar references inthe context of describing the invention (especially in the context ofthe following claims) are to be construed to cover both the singular andthe plural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

1. A compound comprising Formula (I):

wherein R₁₋₆ are independently selected from the group consisting of anelectron withdrawing group, an alkyl group, an aryl group, hydrogen, aheteroaryl group, and a five or six member ring structure formed fromthe R₁ and R₂ pair, the R₃ and R₄ pair, the R₄ and R₅ pair, or the R₅and R₆ pair; R₇ is a substituted or unsubstituted aryl group; and Y isan oxime forming nucleophile.
 2. The compound of claim 1, wherein theoxime forming nucleophile is aminooxy.
 3. The compound of claim 1,wherein the oxime forming nucleophile is hydrazine.
 4. The compound ofclaim 1, wherein the electron withdrawing group is independentlyselected from the group consisting of halogen, NO₂, SO₃R_(S),SO₂N(R_(N))₂, CN, CNS, keto, and alkoxy groups; wherein R_(N) and R_(S)are independently selected from the group consisting of C₁-C₁₀ alkylgroups, which are branched or unbranched, saturated or unsaturated, andsubstituted or unsubstituted, and aryl groups, which are substituted orunsubstituted.
 5. The compound of claim 1, wherein at least one of R₁₋₆is an electron withdrawing group.
 6. The compound of claim 1, wherein atleast one of R₁₋₆ is an alkyl group.
 7. The compound of claim 6, whereinthe alkyl group has from one to ten carbon atoms.
 8. The compound ofclaim 1, wherein at least one of R₁₋₆ is an aryl group.
 9. The compoundof claim 1, wherein at least one of R₁₋₆ is a heteroaryl group.
 10. Thecompound of claim 1, wherein at least one of R₁₋₆ is a hydrogen.
 11. Thecompound of claim 1, wherein at least one of the R₁ and R₂ pair, the R₃and R₄ pair, the R₄ and R₅ pair, or the R₅ and R₆ pair are combined toform a ring having five or six ring members.
 12. The compound of claim11, wherein the ring has six ring members.
 13. The compound of claim 1,wherein R₇ is an aryl group.
 14. The compound of claim 13, wherein thearyl group is selected from the group consisting of phenyl, naphthyl,xylyl, tolyl, pyridyl, and anilinyl.
 15. The compound of claim 14,wherein the aryl group is an anilinyl group.
 16. The compound of claim15, wherein the anilinyl group comprises:

wherein L and L′ are independently selected from the group consisting ofsubstituted or unsubstituted C₁₋₁₀ alkyl, wherein the C₁₋₁₀ alkyl groupsare saturated or unsaturated; and wherein one of L or L′ is substitutedwith an oxime forming nucleophile substituent.
 17. A compoundcomprising:


18. A compound comprising:

wherein R is an alkyl group, B is a nucleobase, and W is selected fromthe group consisting of a protecting group, an oligonucleotide, anucleotide or a nucleoside.
 19. The composition of claim 18, wherein thenucleobase is selected from the group consisting of adenine, guanine,cytosine, uracil and thymine.
 20. The composition of claim 19, whereinthe nucleobase is uracil.
 21. A compound comprising Formula (I):

wherein R₁₋₆ are independently selected from the group consisting of anelectron withdrawing group, an alkyl group, an aryl group, hydrogen, aheteroaryl group, and a five or six member ring structure formed fromthe R₁ and R₂ pair, the R₃ and R₄ pair, the R₄ and R₅ pair, or the R₅and R₆ pair, and wherein at least one of R₁₋₆ is an alkyl group, an arylgroup or a heteroaryl group; R₇ is a substituted or unsubstituted arylgroup; and Y is a nucleophile.
 22. A compound comprising Formula (I):

wherein R₁₋₆ are independently selected from the group consisting of anelectron withdrawing group, an alkyl group, an aryl group, hydrogen, aheteroaryl group, and a five or six member ring structure formed fromthe R₁ and R₂ pair, the R₃ and R₄ pair, the R₄ and R₅ pair, or the R₅and R₆ pair, and wherein at least one of the R₁ and R₂ pair, the R₃ andR₄ pair, the R₄ and R₅ pair, or the R₅ and R₆ pair are combined to forma ring having five or six ring members; R₇ is a substituted orunsubstituted aryl group; and Y is a nucleophile.
 23. The compound ofclaim 22, wherein the ring has six ring members.
 24. A compoundcomprising Formula (I):

wherein R₁₋₆ are independently selected from the group consisting of anelectron withdrawing group, an alkyl group, an aryl group, hydrogen, aheteroaryl group, and a five or six member ring structure formed fromthe R₁ and R₂ pair, the R₃ and R₄ pair, the R₄ and R₅ pair, or the R₅and R₆ pair; R₇ is an anilinyl group; and Y is a nucleophile.
 25. Thecompound of claim 24, wherein the anilinyl group comprises:

wherein L and L′ are independently selected from the group consisting ofsubstituted or unsubstituted C₁₋₁₀ alkyl, wherein the C₁₋₁₀ alkyl groupsare saturated or unsaturated; and wherein one of L or L′ is substitutedwith an oxime forming nucleophile substituent.